ABSTRACT: RNA-seq analysis of five retinoblastoma tumor samples to compare expression signature with retinal organoids generated from hESCs in vitro and modelling retinoblastoma
Project description:Mitochondria play a crucial role in the differentiation and maturation of human cardiomyocytes (CMs). To identify mitochondrial pathways and regulators that are involved in cardiac differentiation and maturation, we examined human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Proteomic analysis was performed on enriched mitochondrial protein extracts isolated from hiPSC-CMs differentiated from dermal fibroblasts (dFCM) and cardiac fibroblasts (cFCM), at different days of differentiation (between 12 and 115 days), and also from adult and neonatal mouse hearts for comparison. Mitochondrial proteins with a ≥2-fold change between differentiation time points in dFCMs and cFCMs, and between adult versus neonatal mouse hearts, were subjected to Ingenuity Pathway Analysis (IPA), and some upregulated proteins were validated by immunoblotting. The highest significant upregulation was in metabolic pathways for fatty acid oxidation (FAO), the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS) and branched chain amino acid (BCAA) catabolism. The top upstream regulators predicted by IPA were- peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1-a), the insulin receptor and the retinoblastoma protein (Rb) transcriptional repressor. In addition, IPA and immunoblotting showed substantial upregulation of the mitochondrial LonP1 protease, which regulates mitochondrial proteostasis, energetics and metabolism. Using this proteomics approach, we have identified key metabolic and intracellular signaling pathways that are up- and down- regulated during the biogenesis of mitochondria in differentiating and maturing cardiac myocytes.
Project description:We performed a high throughput comparative proteomics study to identify differentially expressed proteins that could serve as putative targets in retinoblastoma. We used iTRAQ based quantitative proteomics approach and analyzed the samples on orbitrap velos mass spectrometer. We identified and quantified a total of 3733 proteins in retinoblastoma when compared with normal adult retina. In total, we identified 987 proteins that were differentially expressed in retinoblastoma with a fold change of ≥2 of which 392 proteins were upregulated and 595 were downregulated.
Project description:Transcriptome analysis of partially degraded and fragmented RNA samples from retinoblastoma primary tumors. Global gene expression profiling has shown great promise in high-throughput biomarker discovery for early disease detection in Retinoblastoma tumors. In this analysis we have used 3 retinoblastoma primary tumors in triplicates which was normalised against human healthy Retina. Here, we have used nanogram scale of retinoblastoma RNA, processed in HUMAN GENE 1.0 ST ARRAYS. We have analysed the gene expression in 2 normal healthy adult retina collected from cadaveric eyes and 3 retinoblastoma primary tumors
Project description:Retinoblastoma (RB) is a malignant intraocular neoplasm occurs mostly in children. The malignancy caused due to altered miRNA expression in cancer tumors and circulating body fluids has been reported in several cancers. The aberrantly expressed microRNAs are useful for diagnosis and prognosticating the tumors. Circulating miRNAs have been identified as potential markers in neoplastic and non -neoplastic diseases. The aim of the study involves identifying microRNA signatures of serum of Retinoblastoma and possible use of differential miRNA as unique biomarker for RB. Agilent Single Color Whole miRNA microarray: 14 advanced stage Retinoblastoma Serum (pooled) Vs 14 Non-Retinoblastoma Serum (pooled) samples
Project description:Gene expression profiles in retinoblastoma by whole Human Genome DNA microarrays in comparison to the normal retina gave an insight into several genes and pathways that were regulated in the tumor. The upregulated and the downregulated genes were further validated by quantitative real time PCR and tissue microarray (TMA) on retinoblastoma tumors. Theoverall goal is to determine the global gene expression profile in retinoblastoma tumors which is first of its kind on a whole genome microarray. Two coloured microarray:retinoblastoma tumor (4 samples) vs. normal retina (4 samples).
Project description:Retinoblastoma Y79 cell line was grown on 3D scaffolds and compared its gene expression profile with Y79 cells grown without scaffold. Agilent one-color experiment, Organism: Human, Agilent-014850 Whole Human Genome Microarray 4x44K G4112F, Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
Project description:The retinoblastoma (Rb) protein is a potent tumor suppressor which is known to negatively regulate the cell cycle as well as tumor progression. Phosphorylated Rb protein (pRb) has been demonstrated to be in-charge for the key G1 checkpoint, blocking entry into S-phase and thereby the cell growth. This study was designed to capture interacting protein partners of Rb1 as the cell cycle progresses. Rb1 expressing HEK-293 cells were cultured in light, medium and heavy SILAC labels to capture the changes in Rb1 interactome as the cell cycle progressed from G0 to G1S and then to G2 phase, respectively. This data might help in understanding the cell cycle regulatory effect of Rb1 protein and complement the available information on its interacting partners.
Project description:hESC lines carrying deleterious mutations in the RB1 gene in heterozygous and homozygous state were generated by genome-editing based on CRISPR/Cas9. Parental cell line and genome-edited cell lines were differentiated into retinal organoids for 152 days based on the Protocol published by Döpper et al., Current protocols, PMID: 32956559. Briefly, single cells were reaggregated in presence of dual SMAD and WNT-inhibition; retinal tissue became visible from day 12 onward. BMP4-induction and addition of small molecules CHIR99021 and SU5402 directed differentiation towards retina and retinal pigment epithelium. Long-term differentiation was carried out in the presence of 10% FBS, taurine and retinoic acids. Organoids were collected at indicated time points and either embedded for cryosectioning and immunostaining or frozen at -80°C for RNA preparation.
Project description:Retinoblastoma Y79 cell line was treated with specific siRNA to silence EpCAM gene expression in vitro and RNA was isolated for microarray experiment to study the changes in the whole gene expression profiling in the Y79 cell line. The study identified 465 up-regulated genes (>1.0 fold) and 205 down-regulated genes (<0.5 fold) in response to knockdown of Ep-CAM. Organism used: Homo Sapiens ï Slides: Whole genome Human 4x44k array ï Starting material: Cells in RNA Later ï RNA Samples used: Control, siRNA1 ï Labeling kit: Agilent Low Input RNA Linear amplification kit ï Labeling Method: T7 promoter based-linear amplification to generate labelled complementary RNA ï Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit ï Hybridization Kit: Agilent Insitu Hybridization Kit Hybridization protocol The fragmented cRNA were mixed with 25ul of 2x GE Hybridization Buffer (Agilent). About 45ul of the resulting mixture was applied to the Microarray and hybridized at 65degC for 17 hours in an Agilent Microarray Hybridization Chamber (SureHyb: G2534A) with Hybridization Oven. After hybridization, slides were washed with Agilent Gene expression Wash Buffer I for 1 minute at room temperature followed by a 1 min wash with Agilent Gene expression Wash Buffer II for 37C. Slides were finally rinsed with Acetonitrile for cleaning up and drying. Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Microarrays were scanned on an Agilent scanner (G2565AA) at 100% laser power, 30% PMT.Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturerâs protocol. Data processing Feature extracted data was analyzed using GeneSpring GX v 7.3.1 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended one color Per Chip and Per Gene Data Transformation: Set measurements less than 0.01 to 0.01, Per Chip: Normalize to 50th percentile, Per Gene: Normalize to Specific Samples. Further quality control of normalized data was done using correlation based condition tree to eliminate bad experiments. Differentially regulated Genes were filtered with cutoff of > 1.5 for Up regulation and < 0.55 for Down Regulation were obtained. Differentially regulated genes were clustered using gene tree to identify significant gene expression patterns.
Project description:Retinoblastoma Y79 cell line was treated with scopoletin in vitro and RNA was isolated for microarray experiment to study the changes in the whole gene expression profiling in the Y79 cell line. The study identified 1675 up-regulated genes (>1.0 fold) and 1879 down-regulated genes (<-1 fold) in response to scopoletin treatment Slides:Whole genome Human 4x44k array ï Starting material: Cells in RNA Later ï RNA Samples used: Y79_c_Noni Extract,Control ï Labeling kit: Agilentâs Quick-Amp labeling Kit ï Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA ï Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit Cat ï Hybridization Kit: Agilentâs In situ Hybridzation kit Hybridization protocol The fragmented cRNA were mixed with 25ul of 2x GE Hybridization Buffer (Agilent). About 45ul of the resulting mixture was applied to the Microarray and hybridized at 65degC for 17 hours in an Agilent Microarray Hybridization Chamber (SureHyb: G2534A) with Hybridization Oven. After hybridization, slides were washed with Agilent Gene expression Wash Buffer I for 1 minute at room temperature followed by a 1 min wash with Agilent Gene expression Wash Buffer II for 37C. Slides were finally rinsed with Acetonitrile for cleaning up and drying. Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Microarrays were scanned on an Agilent scanner (G2565AA) at 100% laser power, 30% PMT.Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturerâs protocol. Data processing Feature extracted data was analyzed using GeneSpring GX v 7.3.1 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended one color Per Chip and Per Gene Data Transformation: Set measurements less than 0.01 to 0.01, Per Chip: Normalize to 50th percentile, Per Gene: Normalize to Specific Samples. Further quality control of normalized data was done using correlation based condition tree to eliminate bad experiments. Differentially regulated Genes were filtered with cutoff of > 1.5 for Up regulation and < 0.55 for Down Regulation were obtained. Differentially regulated genes were clustered using gene tree to identify significant gene expression patterns.